title: Development of a PCR assay for rapid detection of Cronobacter spp. from food.
abstract: The occurrence of outbreaks of necrotizing meningitis caused by Cronobacter spp. in neonates highlights the need for rapid detection and accurate identification of this pathogenic species. The gold standard for isolation and identification of Cronobacter spp. from powdered infant formula is time consuming and labor intensive. The gyrB gene that encodes the B subunit of DNA gyrase (topoisomerase type II) was found to be suitable for the identification of Cronobacter spp. A region of the gyrB gene of 38 Cronobacter spp. strains and 5 Enterobacter spp. strains was amplified and sequenced, and a pair of primers was designed and synthesized based on the sequence of the gyrB gene. A polymerase chain reaction (PCR) system was developed and optimized to detect Cronobacter spp. The PCR assay amplified a 438 bp DNA product from all 38 Cronobacter spp. strains tested but not from 34 other bacteria. The detection limit was 1.41 pg/PCR (equivalent 282 genomic copies) when the genomic DNA of Cronobacter sakazakii ATCC 29544 was 10-fold diluted. Infant formula powders from 3 different commercial brands were inoculated with strains ATCC 29544 at a level of 56 colony-forming units, and the target fragment were produced after samples were enriched for 6 h at 37 °C. Twenty-five food samples were evaluated by the PCR assay and the conventional method. A PCR product of the expected size was obtained from 3 samples; however, Cronobacter spp. strains were isolated from only 2 samples by the conventional method. This method is a useful tool for rapid identification of Cronobacter spp. in food and potentially environmental samples.
authors.affiliation: a State Key Laboratory of Dairy Biotechnology, Technology Center of Bright Dairy & Food Co., Ltd., 1518 West Jiangchang Road, Shanghai 200436, People's Republic of China.
authors.forename: Wanyi
authors.initials: W
authors.lastname: Chen
authors.forename: Lianzhong
authors.initials: L
authors.lastname: Ai
authors.forename: Jielin
authors.initials: J
authors.lastname: Yang
authors.forename: Jing
authors.initials: J
authors.lastname: Ren
authors.forename: Yunfei
authors.initials: Y
authors.lastname: Li
authors.forename: Benheng
authors.initials: B
authors.lastname: Guo
mesh_terms: D059126:Cronobacter / Q000235:genetics / Q000302:isolation & purification*; D005516:Food Microbiology; D006801:Humans; D007223:Infant; D041943:Infant Formula; D008581:Meningitis / Q000382:microbiology; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity